In this renewal we propose to establish a more mechanistic basis for the roles of the connexins in cell-cell communication in the microvasculature. We will study radial and longitudinal cell-cell communication in the arterioles, emphasizing the idea that the smooth muscle and the endothelial cell are united to form a functional unit via the myoendothelial cell junctions. The application takes advantage of our extensive theoretical and technical developments in the use of calcium and voltage imaging on in vitro and in vivo systems. We will systematically test the idea the linkage of the calcium pools of the smooth muscle and the endothelium confers unique response patterns on the behavior of arterioles. A unique aspect of this proposal is a comparative study of the arterioles from three species known to have quite different arteriolar responses. Using the tools of molecular genetics we have also begun the process of the process of identification of specific molecular contributors to gap junctional communication. We will include experiments on connexin 37 and 40 knockouts and newly developed smooth muscle and endothelial-cell specific knockouts of connexin 43. We seek to answer three broad questions. First, How does cell-cell coupling between smooth muscle and endothelial cell modulate arteriolar reactivity? Second, what are the intercellular pathways that are involved in the conducted vasomotor response? Third, is gap junctional communication physiologically important? Several interrelated hypotheses will be tested. These are: Hypothesis 1 - Myoendothelial coupling of both membrane potential and calcium pools play a critical role in determining local responses to stimulation Hypothesis 2 - Myoendothelial coupling of changes in membrane potential and not changes in the endothelial cell calcium mediate the conducted vasomotor responses Hypothesis 3 - Similar pathways for cell-cell coupling exist in vitro and in vivo Hypothesis 4 - The endothelium functions as a preferential pathway for dye and electrical coupling. Hypothesis 5 - Intercellular communication is mediated by multiple connexins Hypothesis 6- Gap junctional communication plays a role in the establishment of normal vasomotor tone